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University of Southampton Research Repository ePrints Soton Copyright © and Moral Rights for this thesis are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder/s. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holders. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given e.g. AUTHOR (year of submission) "Full thesis title", University of Southampton, name of the University School or Department, PhD Thesis, pagination http://eprints.soton.ac.uk ECOTOXICOLOGY AND ECOPHYSIOLOGY OF MYSIDS, WITH SPECIAL REFERENCE TO COPPER TOXICITY IN Praunus flexuosus by Eva Garnacho A thesis submitted to the University of Southampton for the degree of Doctor of Philosophy School of Ocean and Earth Sciences Faculty of Science January 2000 UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF SCIENCE SCHOOL OF OCEAN AND EARTH SCIENCE Doctor of Philosophy ECOTOXICOLOGY AND ECOPHYSIOLOGY OF MYSIDS, WITH SPECIAL REFERENCE TO COPPER TOXICITY AND Praunusflexuosus by Eva Garnacho Toxicity of dissolved copper was examined in a common coastal mysid population {Praunusflexuosus). The life cycle and ecophysiology were studied under natural conditions throughout the year, and responses to dissolved copper were determined in the laboratory. Pronounced and ontogenic seasonal differences in copper toxicity for the mysid Praunus flexuosus were observed. Sublethal and lethal parameters (mortality, behaviour, metabolism, reproduction, and bioaccumulation) showed seasonal variation in response to copper toxicity, being highest toxicity in summer. Changes in the form of dissolved copper were measured during toxicity testing, using the chelating resin method to provide a measurement of total and labile (Chelex-available) dissolved in copper the natural seawater used in the toxicity tests. Labile dissolved copper did not show significant variations in the test seawater, confirming that organisms were exposed to constant labile copper concentrations during the toxicity test. The total dissolved copper concentration was significantly higher than the labile form, as organic complexation occurred in natural seawater and during toxicity tests. The labile fraction could be less than 50% of the total fraction. The total dissolved copper concentration decreased significantly when the organism was under stress. While the mortality of the population was insignificant after 10 days of copper exposure (0, 5,25, 75 and 200g 1"') in winter, lethal effects occurred at every copper after exposure level 24 hours (96h LC50 =30.8 g 1"') in summer. The effects of copper on metabolism (respiration and excretion) were very sensitive indicators of sublethal toxicity, which in resulted lethal effects with a prolonged time of exposure. Metabolism shifted to a greater reliance on protein catabolism under copper exposure in both seasons, demonstrating a stronger effect in summer. Total copper content accumulated in the organism increased with increasing copper concentration in solution. Copper accumulation rate was higher in summer than in winter, to rates of . increasing 7^g g*1 dry weight day"1. Reproductive processes were severely disrupted at any copper treatment. Production ofjuveniles was reduced to zero, because of the high abortion rate, reduction on brood survival and damage to fertilisation processes. LIST OF CONTENTS ABSTRACT i LIST OF CONTENTS ii LIST OF TABLES AND FIGURES v ACKNOWLEDGEMENTS vii CHAPTER 1. INTRODUCTION 1 1.1 Mysids (general aspects) 1 1.1.1. Taxonomy 2 1.1.2. Distribution 2 1.1.3. Internal Anatomy 3 1.1.4. Respiration 3 1.1.5. Excretion 3 1.1.6. Blood vascular system 3 1.1.7. Sensory system 4 1.1.8. Reproduction 5 1.1.9. Role in food chain 5 1.1.10. Biochemical composition 7 1.2 Trace metals and copper in the marine environment 7 1.3 Mysids and their use in toxicology 10 1.4 Area of study 12 1.5 Copper in Southampton Water and the Solent 14 1.6 Objectives of the study 16 CHAPTER 2. GENERAL MATERIALS AND METHODS 17 2.1 Sampling methodology for mysids 17 2.2 Aquarium maintenance 18 2.3 Experimental design 19 2.4 Toxicity test 20 2.5 Statistical analysis 21 CHAPTER 3. DISTRIBUTION AND LIFE CYCLE OF Praunusflexuosus IN SOUTHAMPTON WATER AND THE SOLENT 22 3.1 Introduction 22 3.2 The life cycle of Praunusflexuosus 23 3.3 Materials and Methods 24 3.3.1 Distribution of mysid populations 24 3.3.2 Life cycle 24 3.4 Results 25 3.4.1 Distribution of mysids in Southampton Waterand the Solent 25 3.4.2 Life cycle of Praunusflexuosus 29 3.5 Discussion 29 3.6 Summary 33 CHAPTER 4. COPPER SPECIATION AND BIOAVAILABILITY STUDIES 34 4.1. Introduction 34 4.2. Materials and Methods 37 4.2.1. Collection of samples 37 4.2.2. Sample treatment and Chelex extraction of dissolved copper 38 4.2.3. Assessment of accuracy, precision and detection limits of the Chelex technique 39 4.2.4. Storage effects of the sample on the measurement of dissolved copper 39 4.2.5. Measurement of Chelex-available and total copper during toxicity testing 40 4.3 Results 40 4.3.1. Method performance 40 4.3.2. Background concentrations of total dissolved copper in the field and in the.toxicity tests 41 4.3.3. Storage effects on the measurement of total copper of samples .42 4.3.4. Chelex available and total dissolved copper in the toxicity test 44 4.4 Discussion 49 4.5 Summary 53 CHAPTER 5. COPPER TOXICITY IN P.flexuosus 54 5.1 Introduction 54 5.2 Materials and Methods 55 5.2.1 Experimental animals 55 5.2.2 Experimental conditions and design 55 5.2.3 Toxicity tests 56 5.3 Results 57 5.4 Discussion 62 5.5 Summary 66 CHAPTER 6. COPPER BIOACCUMULATION in Praunus flexuosus 67 6.1 Introduction 67 6.2 Materials and Methods 68 6.2.1 Microscope analysis for detection of copper in mysids 68 6.2.2 Analysis of mysids whole body copper content 69 6.3 Results: 70 6.3.1 Microscopy analysis of copper in the mysids 70 6.3.2 Total body copper content in P.flexuosus 71 6.4 Discussion 74 6.5 Summary 78 CHAPTER 7. METABOLISM IN Praunus flexuosus AND EFFECTS OF COPPER EXPOSURE 79 7.1 Introduction 79 7.2 Materials and methods 80 7.2.1 Experimental protocol 80 7.2.2 Metabolism measurements: respiration and excretion 81 7.3 Results 82 7.3.1 Metabolism of the population in winter, spring and summer 82 7.3.2 Metabolic responses to copper 87 7.3.2.1 Oxygen consumption 87 7.3.2.2 Amonia excretion 91 7.3.2.3 O:N ratio 93 7.4 Discussion 96 7.4.1 Routine metabolism 96 7.4.2. Copper toxicity effects 98 7.5 Summary 101 CHAPTER 8. REPRODUCTIVE BIOLOGY OF Praunus flexuosus AND EFFECTS OF COPPER EXPOSURE 102 8.1. Introduction 102 8.1.1 Production of eggs and fertilisation 102 8.1.2 Embryonic development 103 8.1.3 Synchronisation of brooding 103 8.1.4 Marsupium control 103 8.1.5 The reproductive cycle and adaptations to the environment 104 8.1.6 Reproductive strategies in relation to the life cycle 104 8.1.7 Temperature effects 105 8.1.8 Effects of season 106 8.1.8 Pollution effects 106 8.2. Materials and Methods 107 8.2.1 Reproduction under field and laboratory conditions 107 8.2.2 Temperature effects 107 8.2.3 Effects of ambient ammonia, nitrate and nitrite levels on reproduction 108 8.2.4 Effects of copper exposure on reproduction 108 8.2.5 The ability for recovery from copper effects 108 8.3. Results 109 8.3.1. Reproductive biology of Praunus flexuosus under natural conditions 109 8.3.2. Effects of temperature 114 8.3.3. Ammonia, nitrate and nitrite effects on reproduction 116 8.3.4. Copper effects on reproduction 116 8.3.5. Ability to recover from the copper effects 121 8.4. Discussion 124 8.5. Summary 130 CHAPTER 9. GENERAL CONCLUSIONS AND DISCUSSION 131 REFERENCES 135 APPENDICES 149 Appendix 1 150 A. Chelex column extraction method 150 B. Calibration spectrophotometer and pipette 151 C. Experiments results of method performance 152 D. Calculations 154 Appendix II. Data set 155 LIST OF TABLES 1.1. Toxicity tests on mysids 11 1.2. Copper dissolved concentrations ^g l~')in seawater at different locations 15 1.3. Copper concentrations ^g g" ) in marine sediments at different locations 16 3.1. Mysid species distribution in Southampton Water and the Solent 25 3.2. Comparison of the timing of the breeding generations on P.f. at different locations 32 4.1. Detection limits and recovery (%) of copper using the Chelex method 41 4.2. Precision of the Chelex method using different sample treatments 41 4.3. Total copper concentrations of samples from the field (Keyhaven) and from BAS, as used in toxicity tests 42 4.4. Total and Chelex-available copper in the toxicity test 44 LIST OF FIGURES 1.1. Area of study, Southampton Water and the Solent 13 1.2. Effluent discharges into Southampton Water and its effluents 13 3.1. Location of the sampling sites on the study of the distribution of mysids 26 3.2. Variations of temperature at the field site (Keyhaven) 28 3.3. Frequency (%) of the life cycle stages of Praunus flexuosus 30 3.4.